Studies Of Insoluble Proteins Associated With Acute Spinal Cord Injury And Repair In Rat

Acute SCI generally leads to axonal degeneration, neuronal necrosis, glial scar, etc., and causes limb paralysis and paraplegina. After acute SCI, pathological changes occurring in spinal cord are very complex, while the molecular mechanisms involved in those changes are not fully understood. We have to adopt new approach to investigate the complex pathological reactions in injured spinal cord, and proteomics provides us a new pathway.In numerous proteins, insoluble proteins such as membrane or organellar proteins may play important roles in many pathological processes, for example, cell interaction and recognization, ion transport, and signal transduction. In addition, membrane proteins are usually studied as drug targets. Insoluble proteins in spinal cord tissue may also play crucial roles after SCI. By studying these proteins, we could get more theoretical information, which is significant for research. However, because of the extensive protein-protein and protein-lipid interactions in spinal cord, it is very hard to isolate and dissolve these proteins. In order to solve this problem, we used sequential extraction protocol to prepare protein sample, expecting to remove soluble proteins from the sample greatly and enrich the insoluble proteins. Additionally, the composition and the concentration of lysis buffer were modified, so that its efficiency of dissolvation could be promoted.Objective To establish 2-DE gels of insoluble proteins of normal and acute injured rat spinal cord, and to analyze the efficiency of dissolvation and differential expressions of these proteins.Methods By establishing model of injured rat spinal cord and using techniques and theories of proteomics to investigate the insoluble proteins. Protein extraction of spinal cord was gained by using sequential extraction protocol and modified lysis buffer. After CBB R-250 staining and ImageMaster software analysis, MALDI-TOF-MS and database search were used to identify the interest protein spots in 2-DE gels. 5 proteins were randomly selected and submitted to MALDI-TOF/TOF analysis to confirm the reliability of MALDI-TOF-MS results. Parts of the proteins were respectively analyzed by using RT-PCR and Western blot to confirm the reliability of the proteomic analysis. Finally, by using professional software and database, all proteins identified were analyzed in terms of molecular function, biological process, and subcellular localization.Results (1) 2-DE gels of insoluble proteins of the injured rat spinal cord with good reproducibility had been successfully established, and the gels stained by CBB R-250 showed approximately 600 protein spots. (2) After image analysis of 2-DE gels, 62 proteins were found to express differentially. (3) After MALDI-TOF-MS analysis, 44 proteins were identified successfully according to the results of protein database querying. (4) The results of MALDI-TOF/TOF analysis were the same as those of MALDI-TOF-MS identification. (5) The results of RT-PCR and Western blot confirmed the reliability of 2-DE results. (6) The majority of the identified proteins were organellar (70%) and membrane (32%) proteins. The organellar proteins were mainly located in the mitochondrion and nucleus, while the membrane proteins were mainly in plasma membrane, mitochondrial membrane and other membrane. (7) The identified proteins generally belonged to two categories, binder for other molecules (61%) and enzyme catalyzing various reactions (68%). (8) Most of the identified proteins could be divided into five categories, the first was involved in metabolic processes, the second was in developmental processes, the third was in responses to stimulus, the fourth was other proteins, whose roles in spinal cord injury/repair needed further study, and the fifth was those unidentified.Conclusion (1) The insoluble proteins in the sample were obviously enriched, which provides a basis of technology for study in the future. (2) The insoluble proteins were analyzed at four different time points after injury, which made us get more knowledge about the dynamic expression of these proteins. (3) By studying these insoluble proteins, we could get better understanding about the molecular mechanisms of spinal cord injury/repair.